MAP kinase modules are three-kinase enzyme switches comprised of a MAPK, a MAP2K, and a MAP3K. Prior studies have focused primarily on the structures of components of these modules and their activation. Here we will address the nature of the interactions between cascade components and the chemistry of the double phosphorylations catalyzed by MAP2Ks, processes central to the action and retention of MAP kinase cascades. Docking interactions between MAP2Ks and MAPKs, defined in several labs, were studied crystallographically in the low activity forms of MAPKs ERK2 and p38alpha. These data reveal that the docking interactions induce long range conformational changes in the activation loop of the MAPK, 30 A from the docking groove. Since similar coupled changes were observed in both ERK2 and p38alpha, the conformational changes are likely functionally significant, possibly to make the phosphorylation sites available for processing. The first question to be addressed here is whether the same conformation is accessed from the active forms of the MAPKs p38alpha and ERK2. Peptide bound forms of active ERK2 and p38alpha will be solved with existing crystals. Crystals of full-length MEK6-p38alpha complexes will be improved. Docking interactions between MAP3Ks and MAP2Ks have been demonstrated recently. Where is the binding site in the MAP3K? As with the MAP2K- MAPK docking interaction, does the MAP3K-MAP2K interaction involve conformational changes? The MAP3K TAO2, which we have crystallized, will be crystallized in the presence of MEK6-derived peptides. The second question addressed here is how are MAP2Ks capable of carrying out two chemistries, serine/threonine and tyrosine phosphorylation? Are activation loop conformational changes involved in this process as well? A quantitative assay for tyrosine versus threonine phosphorylation of p38alpha by MEK6 has been established that will be used to determine whether the two phosphorylation sites on MEK6, modeled with mutant and partially phosphorylated MEKs, have unique roles in tyrosine versus serine/threonine phosphorylation. Bisubstrate inhibitors, ATP-pTyr- peptide and ATP-pThr-peptide mimetics have been synthesized (by published chemistry) that will be used for crystallographic analysis bound to the MAP2K MEK6.